Multiplex CRISPR/Cas9 editing of gliotoxin biosynthesis genes in Aspergillus fumigatus reduces pathogenicity in broilers.

Cryptic links between apparently unrelated metabolic systems represent potential new drug targets in fungi. Evidence of such a link between zinc and gliotoxin (GT) biosynthesis in Aspergillus fumigatus is emerging. Expression of some genes of the GT biosynthetic gene cluster gli is influenced by the zinc-dependent transcription activator ZafA, zinc may relieve GT-mediated fungal growth inhibition and, surprisingly, GT biosynthesis is influenced by zinc availability. In A. fumigatus, dithiol gliotoxin (DTG), which has zinc-chelating properties, is converted to either GT or bis-dethiobis(methylthio)gliotoxin (BmGT) by oxidoreductase GliT and methyltransferase GtmA, respectively. A double deletion mutant lacking both GliT and GtmA was previously observed to be hypersensitive to exogenous GT exposure. Here we show that compared to wild-type exposure, exogenous GT and the zinc chelator N,N,N′,N′-tetrakis(2-pyridinylmethyl)−1,2-ethanediamine (TPEN) inhibit A. fumigatus ΔgliTΔgtmA growth, specifically under zinc-limiting conditions, which can be reversed by zinc addition. While GT biosynthesis is evident in zinc-depleted medium, addition of zinc (1 µM) suppressed GT and activated BmGT production. In addition, secretion of the unferrated siderophore, triacetylfusarinine C (TAFC), was evident by A. fumigatus wild-type (at >5 µM zinc) and ΔgtmA (at >1 µM zinc) in a low-iron medium. TAFC secretion suggests that differential zinc-sensing between both strains may influence fungal Fe3+ requirement. Label-free quantitative proteomic analysis of both strains under equivalent differential zinc conditions revealed protein abundance alterations in accordance with altered metabolomic observations, in addition to increased GliT abundance in ΔgtmA at 5 µM zinc, compared to wild-type, supporting a zinc-sensing deficiency in the mutant strain. The relative abundance of a range of oxidoreductase- and secondary metabolism-related enzymes was also evident in a zinc- and strain-dependent manner. Overall, we elaborate new linkages between zinc availability, natural product biosynthesis and oxidative stress homeostasis in A. fumigatus.

A substrate-based production system is a simple and low-cost method for arbuscular mycorrhizal (AM) fungal inoculum production. However, it is time-consuming and typically yields low numbers of AM fungal spores due to several factors affecting plant growth efficiency. Our study investigated the use of light-emitting diode (LED) technology to expedite AM fungal spore production in planta. We performed experiments with Rhizophagus irregularis inoculated in maize (Zea mays L.), contrasting LED light with greenhouse (GH) conditions. Our results exhibited a significant improvement in AM fungal colonization and spore production, as well as a reduction in the production period from 120 to 90 days under the LED light condition. This was achieved using a red-and-blue light ratio of 60:40 with a total light intensity of 300 µmol m−2 s−1. The LED light treatments improved maize growth by increasing nitrogen (N) and phosphorus (P) concentrations in shoots and roots, respectively. Our gene expression analyses revealed that in AMF-inoculated plants, genes related to photosynthesis were significantly upregulated under LED light compared to the GH condition. Moreover, LED increased the expression of marker genes linked to the AM fungi-related cell cycle, indicating enhanced AM fungal growth during symbiosis. These findings advance our comprehension of LED applications in agriculture, offering promising prospects for acceleration of AM fungal spore production.

This study evaluated the effects of dietary threonine (Thr) and arginine (Arg) concentrations on growth performance, meat quality, stress indicators, antioxidant capacity, and gut health in broiler chickens raised under multiple stress conditions. A total of 280 21-d-old broiler chickens were randomly allotted to 4 treatments with 8 replicates. The positive control (PC) group was raised under normal rearing conditions (thermoneutral temperature and low stocking density; 22.6 ± 0.6°C for 24 h and 15.2 birds/m<sup>2</sup>), whereas the remaining groups were subjected to multiple stress conditions (cyclic heat stress and high stocking density; 29.3 ± 0.9°C for 10 h, 22.8 ± 0.8°C for 14 h, and 30.3 birds/m<sup>2</sup>). Both the PC and negative control (NC) groups received a basal diet, while the other two groups were provided diets containing 1.3-fold higher concentrations of digestible Thr or Arg compared to the basal diet. Experimental diets and water were supplied ad libitum for 14 d. Results indicated that growth performance was decreased (<italic>p</italic> < 0.05) in NC, Thr, and Arg groups compared to PC group. Birds in NC group had greater (<italic>p</italic> < 0.05) heterophil to lymphocyte ratio and feather corticosterone (CORT) concentrations than those in PC group. Under multiple stress conditions, Thr and Arg groups showed less (<italic>p</italic> < 0.05) feather CORT concentrations than NC group. In hepatic tissue, reactive oxygen species levels were increased (<italic>p</italic> < 0.05) in NC and Arg groups compared to PC group. Jejunal nitric oxide levels were increased (<italic>p</italic> < 0.05) in NC group compared to PC, Thr, and Arg groups. Under multiple stress, Thr and Arg treatment groups exhibited greater (<italic>p</italic> < 0.05) transepithelial electrical resistance values than NC group. The expression levels of <italic>OCLN</italic>, <italic>ZO-1</italic>, and <italic>HSP70</italic> were greater (<italic>p</italic> < 0.05) in Thr and Arg groups than in NC group. In conclusion, increasing dietary concentrations of Thr and Arg improves intestinal integrity and mitigates stress response in broiler chickens exposed to multiple stress.

Gliotoxin (GT) biosynthesis in fungi is encoded by the gli biosynthetic gene cluster. While GT addition autoinduces biosynthesis, Zn2+ has been shown to attenuate cluster activity, and it was speculated that identification of Zn2Cys6 binuclear transcription factor GliZ binding partners might provide insight into this observation. Using the Tet-ON induction system, doxycycline (DOX) presence induced GliZ fusion protein expression in, and recovery of GT biosynthesis by, A. fumigatus ΔgliZ::HA-gliZ and ΔgliZ::TAP-gliZ strains, respectively. Quantitative RT-PCR confirmed that DOX induces gli cluster gene expression (n = 5) in both A. fumigatus HA-GliZ and TAP-GliZ strains. GT biosynthesis was evident in Czapek-Dox and in Sabouraud media, however tagged GliZ protein expression was more readily detected in Sabouraud media. Unexpectedly, Zn2+ was essential for GliZ fusion protein expression in vivo, following 3 h DOX induction. Moreover, HA-GliZ abundance was significantly higher in either DOX/GT or DOX/Zn2+, compared to DOX-only. This suggests that while GT induction is still intact, Zn2+ inhibition of HA-GliZ production in vivo is lost. Co-immunoprecipitation revealed that GT oxidoreductase GliT associates with GliZ in the presence of GT, suggesting a potential protective role. Additional putative HA-GliZ interacting proteins included cystathionine gamma lyase, ribosomal protein L15 and serine hydroxymethyltransferase (SHMT). Total mycelial quantitative proteomic data revealed that GliT and GtmA, as well as several other gli cluster proteins, are increased in abundance or uniquely expressed with GT addition. Proteins involved in sulphur metabolism are also differentially expressed with GT or Zn2+ presence. Overall, we disclose that under DOX induction GliZ functionality is unexpectedly evident in zinc-replete media, subject to GT induction and that GliT appears to associate with GliZ, potentially to prevent dithiol gliotoxin (DTG)-mediated GliZ inactivation by zinc ejection.

Larvae of saprophagous insects often have been suspected of being competitors of filamentous fungi on decaying organic matter, which negatively influence mold development. Of interest, the role of insects in determining fungal growth and the onset of sporulation largely has been ignored. I used Aspergillus niger and the vinegar fly Drosophila melanogaster as an ecological model system to analyze the influence of insect larvae on daily fungal growth and the start of conidiospore production. I used an artificial substrate to test whether the effect of larval density (one, five and 10 larvae) and inoculation date of the mold (2 and 3 d ahead of the addition of larvae) significantly altered fungal growth. Fungal growth (area covered by hyphal tissue of the artificial patch) was affected negatively by the number of larvae and by the time that elapsed between inoculation with fungal spores and transfer of larvae to the patches. Whereas one larva had only a minor effect on fungal growth, five or 10 larvae strongly hampered mold development. As time between inoculation with spores and introduction of fly larvae increased, mold increased, indicating a priority effect for the fungus. When 10 larvae were transferred at the same time as the patches were inoculated with spores, almost no mold was visible within the period of observation (after 12 d). In comparison with control treatment (no insect larvae), an increase in larval density caused an increasing delay of several days in the start of spore production. Thus only minor changes in the density of insect larvae and the time that larvae entered the patches after inoculation with spores had an enormous effect on fungal growth and spore production. Therefore insects co-occurring with mold on ephemeral resources might constitute an important biotic factor driving local fungal population dynamics. The mechanisms leading to the suppression of fungal growth and the evolutionary implications of insect-mold interactions are discussed.

Larvae of saprophagous insects often have been suspected of being competitors of filamentous fungi on decaying organic matter, which negatively influence mold development. Of interest, the role of insects in determining fungal growth and the onset of sporulation largely has been ignored. I used Aspergillus niger and the vinegar fly Drosophila melanogaster as an ecological model system to analyze the influence of insect larvae on daily fungal growth and the start of conidiospore production. I used an artificial substrate to test whether the effect of larval density (one, five and 10 larvae) and inoculation date of the mold (2 and 3 d ahead of the addition of larvae) significantly altered fungal growth. Fungal growth (area covered by hyphal tissue of the artificial patch) was affected negatively by the number of larvae and by the time that elapsed between inoculation with fungal spores and transfer of larvae to the patches. Whereas one larva had only a minor effect on fungal growth, five or 10 larvae strongly hampered mold development. As time between inoculation with spores and introduction of fly larvae increased, mold increased, indicating a priority effect for the fungus. When 10 larvae were transferred at the same time as the patches were inoculated with spores, almost no mold was visible within the period of observation (after 12 d). In comparison with control treatment (no insect larvae), an increase in larval density caused an increasing delay of several days in the start of spore production. Thus only minor changes in the density of insect larvae and the time that larvae entered the patches after inoculation with spores had an enormous effect on fungal growth and spore production. Therefore insects co-occurring with mold on ephemeral resources might constitute an important biotic factor driving local fungal population dynamics. The mechanisms leading to the suppression of fungal growth and the evolutionary implications of insect-mold interactions are discussed.

Comparative efficacy of a phytogenic feed additive and an antibiotic growth promoter on production performance, caecal microbial population and humoral immune response of broiler chickens inoculated with enteric pathogens.

The impact of climate change on fungal growth and spore production is less well documented than for allergenic pollen grains, although similar implications for respiratory tract diseases in humans occur. Fungal spores are commonly described as either “dry” or “wet” according to the type of weather associated with their occurrence in the air. This study examined the distribution of selected fungal spores (Alternaria spp., Cladosporium spp., Didymella spp., Epicoccum spp., Leptosphaeria spp. and rusts) occurring in the West Midlands of UK during 2 years of contrasting weather. Spore specimens were collected using a 7-day volumetric air sampler and then analysed with the aid of light microscopy. Distributions of spores were then studied using normality tests and Mann–Whitney U test, while relationships with meteorological parameters were investigated using Spearman’s rank test and angular-linear correlation for wind direction analysis. Our results showed that so-called wet spores were more sensitive to the weather changes showing statistically significant differences between the 2 years of study, in contrast to “dry” spores. We predict that in following years we will observe accelerated levels in allergenic fungal spore production as well as changes in species diversity. This study could be a starting point to revise the grouping system of fungal spores as either “dry” or “wet” types and their response to climate change

The objective of this study was to investigate the effects of dietary threonine and arginine levels on stress response, animal welfare score, antioxidant capacity, and jejunal morphology of broiler chickens raised under multiple stress conditions. A total of 280 21-d-old Arbor Acres broiler chickens were randomly allotted to 4 treatments with 8 replicates. Birds were raised under normal conditions (22.6 ± 0.6°C and 15.2 birds/m2) and multiple stress conditions (cyclic heat stress and high stocking density; 29.3 ± 0.9°C for 10 h, 22.8 ± 0.8°C for 14 h, and 30.3 birds/m2). Birds in positive control (PC) were raised under normal conditions, while the other groups were exposed to multiple stress conditions. A basal diet was assigned to PC and negative control (NC). Two additional diets were formulated to contain 1.3-fold higher concentrations of digestible threonine (Thr) or arginine (Arg) than the basal diet. The experimental diets and water were provided ad libitum for 14 d. Animal welfare score including gait score, footpad dermatitis, hock burn, and feather cleanliness was analyzed according to Welfare Quality® assessment protocol for poultry. To analyze stress response, antioxidant capacity, and jejunal morphology, 1 bird per replicate was euthanized by CO2 inhalation. All data were analyzed by ANOVA using the PROC MIXED procedure of SAS (SAS Institute Inc., NC, USA). The replicate was considered an experimental unit. Significance for statistical tests was considered at P < 0.05. Results indicated that heterophil to lymphocyte ratio was less (P < 0.05) in PC group than in other groups. Birds in PC, Thr, and Arg groups had less (P < 0.05) feather corticosterone concentrations than those in NC group. For the animal welfare score, gait score, footpad dermatitis, hock burn, and feather cleanliness were not affected by dietary treatments and multiple stress conditions. Reactive oxygen species (ROS) in the liver were less (P < 0.05) in PC and Arg groups than in NC group. Birds in PC, Thr, and Arg groups had less (P < 0.05) nitric oxide (NO) values in the jejunum than in NC group. Villus height was greater (P < 0.05) in PC group than in NC, Thr, and Arg groups. Birds in PC group had greater (P < 0.05) goblet cell counts than in NC and Arg groups. In conclusion, multiple stress conditions increase the stress response and damage the liver and jejunum of broiler chickens. Increasing concentrations of digestible Thr and Arg in diets reduce stress response, ROS, and NO levels of broiler chickens exposed to multiple stress conditions.

An experiment was conducted to investigate the effects of replacing antibiotic growth promoter (AGP) with a combination of essential oil and organic acids (EO + OA) on growth performance, gut microbiota and immune response in broiler chickens. In Experiment 1, 320 day-old broiler chicks were randomly distributed to 32 pens with 10 birds in each pen and the pens were equally allotted to four treatment groups. In Experiment 2, 120 day-old chicks were divided into the same four treatment groups, each group containing three replicated pens with 10 birds in each pen. The groups were (1) Negative Control (NC) without AGP or other growth-promoting feed additives; (2) AGP (NC + enramycin; 125 mg/kg feed; (3) OA (NC + OA; 500 mg/kg feed); and (4) EO + OA (NC + a combination of cinnamaldehyde and calcium formate; 500 mg/kg feed). Experiment 1 lasted for 40 days, whereas Experiment 2 continued for 28 days. In Experiment 2, all birds were orally challenged with Escherichia coli (108 bacteria/bird) on Day 14. Overall intake, growth and feed conversion ratio (FCR) on Day 40 had no difference (P > 0.05) among the groups in Experiment 1. In Experiment 2, growth, feed intake and FCR were not affected by any dietary treatments until Day 14, but after being challenged with E. coli, bodyweight gain and FCR improved (P < 0.05) for AGP and EO + OA compared with NC. Mortality rate was also lower (P < 0.05) for AGP and EO + OA than NC in Experiment 2. Villi height was higher (P < 0.001) in OA and EO + OA groups compared with NC and AGP groups. Any treatment did not affect (P > 0.05) the counts of total bacteria, E. coli and Lactobacillus in the contents of ileum and caecum. However, Salmonella counts in the ileal and caecal contents decreased (P < 0.001) for AGP, OA and EO + OA compared with NC group. Clostridium counts were lower for EO + OA group than for NC and AGP groups in the ileum, and for AGP, OA and EO + OA groups than for NC in the caeca (P < 0.05). Antibody titer on Day 35 against Newcastle disease vaccine was higher in EO + OA group than in NC, AGP and OA groups (P < 0.001). In conclusion, EO + OA did not affect growth and FCR in broilers. However, AGP and EO + AO improved growth performance and FCR after being challenged with E. coli. Moreover, EO + OA was effective in reducing the Clostridium count in the small intestine and caecum and increasing the villus height and antibody titer against Newcastle disease vaccine.

BackgroundAspergillus fumigatus produces a number of secondary metabolites, one of which, gliotoxin, has been shown to exhibit anti-fungal activity. Thus, A. fumigatus must be able to protect itself against gliotoxin. Indeed one of the genes in the gliotoxin biosynthetic gene cluster in A. fumigatus, gliT, is required for self-protection against the toxin- however the global self-protection mechanism deployed is unclear. RNA-seq was employed to identify genes differentially regulated upon exposure to gliotoxin in A. fumigatus wild-type and A. fumigatus ∆gliT, a strain that is hypersensitive to gliotoxin.ResultsDeletion of A. fumigatus gliT resulted in altered expression of 208 genes (log2 fold change of 1.5) when compared to A. fumigatus wild-type, of which 175 genes were up-regulated and 33 genes were down-regulated. Expression of 164 genes was differentially regulated (log2 fold change of 1.5) in A. fumigatus wild-type when exposed to gliotoxin, consisting of 101 genes with up-regulated expression and 63 genes with down-regulated expression. Interestingly, a much larger number of genes, 1700, were found to be differentially regulated (log2 fold change of 1.5) in A. fumigatus ∆gliT when challenged with gliotoxin. These consisted of 508 genes with up-regulated expression, and 1192 genes with down-regulated expression. Functional Catalogue (FunCat) classification of differentially regulated genes revealed an enrichment of genes involved in both primary metabolic functions and secondary metabolism. Specifically, genes involved in gliotoxin biosynthesis, helvolic acid biosynthesis, siderophore-iron transport genes and also nitrogen metabolism genes and ribosome biogenesis genes underwent altered expression. It was confirmed that gliotoxin biosynthesis is induced upon exposure to exogenous gliotoxin, production of unrelated secondary metabolites is attenuated in A. fumigatus ∆gliT, while quantitative proteomic analysis confirmed disrupted translation in A. fumigatus ∆gliT challenged with exogenous gliotoxin.ConclusionsThis study presents the first global investigation of the transcriptional response to exogenous gliotoxin in A. fumigatus wild-type and the hyper-sensitive strain, ∆gliT. Our data highlight the global and extensive affects of exogenous gliotoxin on a sensitive strain devoid of a self-protection mechanism and infer that GliT functionality is required for the optimal biosynthesis of selected secondary metabolites in A. fumigatus.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-894) contains supplementary material, which is available to authorized users.

Aspergillus fumigatus is an opportunistic fungal pathogen that secretes an array of immune-modulatory molecules, including secondary metabolites (SMs), which contribute to enhancing fungal fitness and growth within the mammalian host. Gliotoxin (GT) is a SM that interferes with the function and recruitment of innate immune cells, which are essential for eliminating A. fumigatus during invasive infections. We identified a C6 Zn cluster-type transcription factor (TF), subsequently named RglT, important for A. fumigatus oxidative stress resistance, GT biosynthesis and self-protection. RglT regulates the expression of several gli genes of the GT biosynthetic gene cluster, including the oxidoreductase-encoding gene gliT, by directly binding to their respective promoter regions. Subsequently, RglT was shown to be important for virulence in a chemotherapeutic murine model of invasive pulmonary aspergillosis (IPA). Homologues of RglT and GliT are present in eurotiomycete and sordariomycete fungi, including the non-GT-producing fungus A. nidulans, where a conservation of function was described. Phylogenetically informed model testing led to an evolutionary scenario in which the GliT-based resistance mechanism is ancestral and RglT-mediated regulation of GliT occurred subsequently. In conclusion, this work describes the function of a previously uncharacterised TF in oxidative stress resistance, GT biosynthesis and self-protection in both GT-producing and non-producing Aspergillus species.

Aspergillus fumigatus is an opportunistic fungal pathogen that secretes an array of immune-modulatory molecules, including secondary metabolites (SMs), which contribute to enhancing fungal fitness and growth within the mammalian host. Gliotoxin (GT) is a SM that interferes with the function and recruitment of innate immune cells, which are essential for eliminating A. fumigatus during invasive infections. We identified a C6 Zn cluster-type transcription factor (TF), subsequently named RglT, important for A. fumigatus oxidative stress resistance, GT biosynthesis and self-protection. RglT regulates the expression of several gli genes of the GT biosynthetic gene cluster, including the oxidoreductase-encoding gene gliT, by directly binding to their respective promoter regions. Subsequently, RglT was shown to be important for virulence in a chemotherapeutic murine model of invasive pulmonary aspergillosis (IPA). Homologues of RglT and GliT are present in eurotiomycete and sordariomycete fungi, including the non-GT-producing fungus A. nidulans, where a conservation of function was described. Phylogenetically informed model testing led to an evolutionary scenario in which the GliT-based resistance mechanism is ancestral and RglT-mediated regulation of GliT occurred subsequently. In conclusion, this work describes the function of a previously uncharacterised TF in oxidative stress resistance, GT biosynthesis and self-protection in both GT-producing and non-producing Aspergillus species.

The influence of collembolans and earthworms on AM fungal mycelium

Genetic control of asexual development in aspergillus fumigatus.